Osteopontin (OSP) has been implicated in several models of tubulointerstitial injury, including chronic cyclosporine (CSA) nephropathy. OSP expression increases in the areas of tubular injury, and. co-localizes with influxing macrophages in the areas of interstitial fibrosis. Thus, these observations suggest that OSP may play a role in the pathogenesis of subacute forms of renal injury. However, because these data are correlative, the precise molecular role of OSP, and whether it exacts beneficial or detrimental effects to the injured renal tissue are unknown. Therefore, the purpose of this application is to develop an in vitro model in which the molecular consequences of OSP expression by proximal tubule epithelium can be defined. To this end, we propose to establish OSP positive (OSP+) and negative (OSP-) clones of the recently developed human proximal tubule cell line, HK-2. Cells that differ only in the expression of OSP will be used comparatively to define the role of OSP in attraCting, binding, and inducing gene expression in populations of macrophages and lymphocytes. Once established, the model will also be used to determine whether OSP expression alters susceptibility to injury and repair. The following specific aims are proposed: i) Define the molecular variant of OSP expressed by HK-2 cells- determine its level of expression, and discern whether its expression can be regulated by appropriate biological response modifiers; 2) Establish OSP(-) HK-2 cells by transient inhibition of OSP expression using phosphorothioate oligonucleotides complementary to OSP mRNA, and\or by transducing genetic suppressor elements specific for the OSP gene; 3) OSP( +) and (-) HK-2 cells will be used comparatively to study binding of macrophages and lymphocytes, and to determine the ability of OSP to induce expression of known inflammatory response modifiers and cytokines; and 4) Determine whether the OSP+ and OSP- HK-2 cells differ in their response to injury and the ability to proliferate in the aftermath of it. The studies proposed in this application are based on the hypothesis that defining the function of a specific protein in a complex tissue system can be greatly facilitated by creating a comparative system in which the only variable is the presence or absence of the protein of interest. By establishing the HK-2 OSP+ and OSP- variants, it should be possible to determine the function of OSP as expressed by HK-2 cells. If binding or signaling functions can be attributed to OSP, it will then be possible in future studies to identify the domains responsible for these activities, and develop reagents that can specifically block them.